Vaccinia DNA Replication

描述（由申请人提供）：遗传物质的忠实复制可以说是所有生物过程中最基本的。对于DNA病毒和细胞一样，一个复杂的蛋白质库介导DNA合成，并协调复制与重组和修复。在这个建议中，我们提出了我们的计划，继续调查如何牛痘病毒，原型痘病毒，完成基因组复制。痘病毒是唯一的DNA病毒之一，其感染周期仅限于受感染细胞的细胞质。这种独立于宿主细胞核的自主性带来了不寻常的挑战，因为病毒必须编码一个完整的复制装置，并建立专门的细胞质复制位点。痘病毒除了具有重要的生物医学意义之外，还代表了有趣的模型系统。我们的研究的影响进一步加强了病毒复制蛋白作为合理的抗病毒治疗的发展目标的重要性。在下一个资助期内，我们将利用遗传学、生物化学和细胞生物学方法，通过以下三个目标进一步阐明牛痘病毒的复制：AIM I：分析复制和修复中的E9/A20/UDG聚合酶全酶。我们将继续研究DNA聚合酶复合物，它由E9 DNA Pol和由A20和D4蛋白组成的异二聚体持续合成因子组成。我们将专注于了解UDG/A20赋予聚合酶持续合成能力的机制。由于D4是一种活性尿嘧啶DNA糖基化酶（UDG），我们将研究病毒和细胞蛋白如何完成复制相关的碱基切除修复。 AIM II：分析DNA复制的机制，并研究D5引物酶/解旋酶、A50连接酶和G5核酸酶对这一过程的贡献。我们将重新探讨痘病毒复制的机制，并评估前导链和滞后链合成的可能参与。在这种情况下，我们将继续我们的分析的基本D5 ATP酶，这是可能有引发酶和解旋酶活性。我们还将研究病毒DNA连接酶和G5蛋白（与FEN 1核酸酶具有同源性）在病毒基因组复制和成熟中的作用。 目的III：分析病毒复制工厂的组装和结构。病毒复制发生在被称为复制工厂的专用细胞质区域。这些工厂由来自内质网（ER）的膜界定并与其相关。我们将测试我们的假设，丰富的H5蛋白作为一个支架内的工厂。此外，我们将使用一个有针对性的siRNA库，以确定细胞膜运输蛋白，有助于重组的ER复制工厂组装的目的。

DESCRIPTION (provided by applicant): The faithful duplication of the genetic material is arguably the most fundamental of all biological processes. For DNA viruses as for cells, a complex repertoire of proteins mediates DNA synthesis and coordinates replication with recombination and repair. In this proposal, we present our plan for continued investigation of how vaccinia virus, the prototypic poxvirus, accomplishes genome replication. Poxviruses are among the only DNA viruses whose infectious cycle is restricted to the cytoplasm of infected cells. This autonomy from the host nucleus poses unusual challenges, in that the virus must encode a complete replication apparatus and establish dedicated cytoplasmic sites for replication. Poxviruses represent intriguing model systems in addition to being of significant biomedical importance. The impact of our studies is further strengthened by the importance of the viral replication proteins as targets for the development of rational antiviral therapies. During the next funding period, we will utilize genetic, biochemical and cell biological approaches to further elucidate the replication of vaccinia virus by focusing on the following three aims: AIM I: Analysis of the E9/A20/UDG polymerase holoenzyme in replication and repair. We will continue our studies of the DNA polymerase complex, which is comprised of the E9 DNA Pol and a heterodimeric processivity factor consisting of the A20 and D4 proteins. We will focus on understanding the mechanism whereby UDG/A20 confers processivity on Pol. Because D4 is an active uracil DNA glycosylase (UDG), we will investigate how replication-associated base excision repair is accomplished by viral and cellular proteins. AIM II: Analysis of the mechanism of DNA replication, and investigation of the contributions of the D5 primase/helicase, the A50 ligase, and the G5 nuclease to this process. We will revisit the mechanism of poxvirus replication and assess the possible involvement of both leading and lagging strand synthesis. In this context, we will continue our analysis of the essential D5 ATPase, which is likely to have both primase and helicase activity. We will also investigate the roles of the viral DNA ligase and the G5 protein, which has homology to FEN1 nucleases, in the replication and maturation of the viral genome. AIM III: Analysis of the assembly and structure of viral replication factories. Viral replication takes place in dedicated cytoplasmic regions known as replication factories. These factories are delimited by, and associated with, membranes derived from the endoplasmic reticulum (ER). We will test our hypothesis that the abundant H5 protein serves as a scaffold within the factories. Furthermore, we will use a targeted siRNA library to identify cellular membrane-trafficking proteins that contribute to the reorganization of the ER for the purpose of replication factory assembly.